In order to maintain their shape and integrity, it is critical that all types of cells contain a structural scaffold. This structure is known as the cytoskeleton and is composed of a framework of interlocking proteins. The major protein component of the cytoskeleton is actin, and the assembly of actin monomers into the cytoskeleton is highly regulated.
Many cellular processes are mediated by the cytoskeleton, especially those involving the interaction of the cell with the surrounding environment. These include cell adhesion, cell motility and cell polarity. For example, changes occurring during cell cycle progression such as those associated with surface adhesion signals are dependent on the appropriate assembly and disassembly of the cytoskeleton. Therefore, it is currently believed that the survival of the cell depends on the controlled regulation of the cytoskeleton.
RhoA, a member of the Rho subfamily of small GTPases, is a protein that has been shown to be involved in a diverse set of signaling pathways including the ultimate regulation of the dynamic organization of the cytoskeleton. The first known biological function of RhoA, described in Swiss 3T3 fibroblasts, was the formation of stress fibers (actin filament bundles) and focal adhesion complexes upon the addition of extracellular ligands (Ridley, Int. J. Biochem. Cell Biol., 1997, 29, 1225-1229). These structures allow the cell to attach and pull along an extracellular substrate altering the cell's shape and position. Since then, the assembly of the cytoskeleton through the activation of RhoA has been demonstrated in epithelial cells, endothelial cells, astrocytes, lymphocytes, preadipocytes, platelets and neurons. While RhoA activation of cytoskeletal assembly most often results in the growth or extension of a cell, in neurons, RhoA has been shown to induce neurite retraction and cause cell rounding, (Hall, Science, 1998, 279, 509-514).
RhoA has also been shown to mediate actin-independent signaling cascades. These include (i) gene expression by activation of the serum response factor (SRF) which, along with ternary complex factors (TCFs), interacts with serum response elements found in certain gene promoters like c-fos, (ii) cell cycle progression through G.sub.1 phase and (iii) induction of tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107).
Manifestations of altered RhoA regulation appear in both injury and disease states. It has been proposed that acute central nervous system trauma may contribute to the development of Alzheimer's disease. Findings that show a high concentration of thrombin, a serine-protease in the blood clotting cascade, localized to the plaques of Alzheimer's disease brains support this claim. An excess of thrombin has been shown to stimulate Rho A activity with a concomitant increase in apoptosis (programmed cell death) (Donovan et al., J. Neurosci., 1997, 17, 5316-5326). These studies also imply a role for RhoA in wound repair and clotting disorders.
RhoA is believed to be involved in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Recent studies demonstrate that RhoA-regulated pathways can induce both changes in cells. Injecting cells transformed with rhoA genes directly into the bloodstream of mice produced metastasis, or tumor growth, in distant organs (del Peso et al., Oncogene, 1997, 15, 3047-3057).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of RhoA. To date, strategies aimed at inhibiting RhoA function have involved the use of bacterial enzymes such as the Clostridium botulinum C3 exoenzyme which ADP ribosylates the protein rendering it inactive or agents (natural enzyme inhibitors or monoterpenes) to inhibit the posttranslational modification (isoprenylation) of RhoA. The use of monoterpenes (plant-derived pyrophosphates) resulted in the reduction of transforming capacity of cancer cells in mammary gland epithelial cells by inactivating RhoA (Ren and Gould, Carcinogenesis, 1998, 19, 827-832). However, these targeting strategies are not specific to RhoA, as many proteins undergo similar posttranslational modifications. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting RhoA function.